Advances in the areas of gene therapy and DNA vaccination have created a need for the large scale manufacture and purification of clinical-grade plasmid DNA. As pointed out in a recent review (Prazeres, et al., 1999, TIBTech 17: 169-174), despite previous work on small scale plasmid DNA purification methodology, it has been difficult to scale up the manufacture and purification of clinical-grade plasmid DNA. Especially problematic have been downstream processing steps, which for the most part have relied on alkaline lysis of the harvested cells, followed by ammonium acetate precipitation and further downstream processing steps relying heavily on size exclusion, anion exchange and reversed phase chromatography steps. In addition, it should be noted that the expense of raw materials, such as resins and buffers, for multiple chromatographic steps become prohibitive due high unit cost and poor capacity for the large DNA molecules. It is known that the cationic detergent CTAB and various forms of silica have been used for the small scale plasmid DNA preparations and not designed to produce clinical grade plasmid vaccine. The ability of these steps to remove certain impurities has not been recognized nor has their utility for scalable process design. Del Sal et al. (1989, BioTechniques 7(5): 514-519) and Gustincich et al. (1991, BioTechniques 11(3): 298-301) use CTAB to precipitate plasmid DNA from clarified small scale E. coli lysates and genomic DNA from small scale preparations of whole human blood, respectively. Ishaq et al. (1990, Biotechniques 9(1): 19-24) disclose the application of small scale CTAB-precipitated plasmid DNA to a PZ523 spin column, yielding a purified product which is at least suitable as a template for subcloning and dideoxy sequencing. None of this art teaches or suggests the use of detergent-based precipitation steps to produce clinical grade lots of DNA plasmid.
Vogelstein & Gillespie (1979, Proc. Natl. Acad. Sci. USA. 76(2): 615-619) disclose a technique for separating restriction enzyme digests of DNA from agarose gels, in which DNA in the presence of concentrated sodium iodide is bound to glass (silica), washed with ethanol, and eluted at a low salt concentration. Boom et al. (1990, J. Clin. Microbiol. 28(3): 495-503) and Carter & Milton (1993, Nucleic Acids Res. 21(4): 1044) disclose methods for the isolation of plasmid DNA which is suitable for DNA sequencing. Plasmid DNA in the presence of the chaotropic agent guanidinium thiocyanate is bound to silica in the form of diatomaceous earth. The immobilized plasmid DNA is washed with ethanol and eluted at low salt concentrations. Subtle variations of this technique are disclosed in (1) PCT Publication WO 91/10331; (2) PCT Publication WO 98/04730, as well as (3) U.S. Pat. No. 5,075,430, issued to Little on Dec. 24, 1999, which discloses a method of isolating plasmid DNA which depends upon adsorption of the DNA onto diatomaceous earth in the presence of a chaotropic agent followed by separation and elution of the DNA; and (4) U.S. Pat. No. 5,808,041, issued to Padhye et al. on Sep. 15, 1998, which discloses a method of nucleic acid isolation utilizing a composition comprising silica gel and glass particles in the presence of a chaotropic agent. Again, these techniques have not been successfully applied to methodology for large scale DNA plasmid preparations required for generation of gram quantities of plasmid DNA for clinical grade formulations for administration to humans and other potential hosts.
U.S. Pat. No. 4,923,978, issued to McCormick on May 8, 1990, discloses the use of silica to purify DNA by preferentially binding proteinaceous materials.
U.S. Pat. No. 5,576,196, issued to Hom et al. on Nov. 19, 1996, discloses the use of silica to purify DNA by preferentially binding RNA.
U.S. Pat. Nos. 5,523,392 and 5,525,319, issued to Woodard et al. on Jun. 4, 1996 and Jun. 11, 1996, respectively, disclose boron silicates, phosphosilicates, and aluminum silicates which can be used as binding surfaces for DNA purification.
PCT International Application PCT/US96/20034 (International publication No. WO 98/01464) discloses the use of hydrated calcium silicate to selectively separate organic compounds from biological fluids, such as blood.
Again, none of the above-identified references provide adequate guidance to the artisan of ordinary skill to provide a methodology to prepare scalable, clinical grade DNA plasmid lots which are substantially free of host cell protein, host cell endotoxin, genomic DNA, genomic RNA and plasmid degradates such as linear and open circle forms. To this end, it would be extremely useful to identify a scaleable plasmid purification process which eliminates the requirement of prohibitively expensive chromatography steps while also providing for gram quantities of a DNA plasmid preparation which is clinical grade for use in at least human vaccination and human gene therapy applications. The present invention addresses and meets these needs by disclosing a scaleable plasmid purification process which preferably utilizes a cationic detergent such as CTAB to selectively precipitate plasmid DNA in an upstream step in combination with downstream large scale batch adsorption steps using hydrated, crystalline calcium silicate (herein, “hcCaSiO3”) or any similar acting compound to remove remaining contaminants such as genomic DNA, genomic RNA, protein, host endotoxin and plasmid degradates such as linear and open circle forms.